The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays an important role in the development and spread of cancer throughout the body. EGFR, also known as erb-b1, is a member of a family of four genes that also includes HER2/neu (erb-b2), HER3 (erb-b3) and HER4 (erb-b4). EGFR is expressed in a wide range of solid tumors, including colon cancers, head and neck cancers, pancreatic cancers, ovarian cancers, and breast cancers.
HER2/neu is a cell surface receptor protein with tyrosine kinase activity. The complete protein consists of three parts: an intracellular cytoplasmic domain, a short hydrophobic transmembrane segment and an extracellular domain (ECD) that is responsible for ligand binding. This receptor protein is expressed on the cell membrane of a variety of epithelial cell types and, through binding of specific growth factors, regulates various aspects of cell growth division.
Her2/neu, the gene that encodes for the HER2/neu protein, is a member of a group of genes known as proto-oncogenes. Proto-oncogenes encode important proteins, such as growth factors, growth factor receptors, and apoptotic proteins, that are involved in normal cell growth and differentiation. When proto-oncogenes are altered by point mutation, translocation or gene amplification, they produce growth signals that may lead to aberrant cellular transformation and the development of cancer.
While Her2/neu can be expressed at low levels in many normal cells, it is typically overexpressed in a variety of cancers. Overexpression of Her2/neu is caused in most cases by an increase in copy number of the gene (gene amplification) and/or by an increase in expression level of the Her2/neu genes in the cell. Overexpression of this growth factor receptor plays a key role in tumor progression by causing a higher rate of cell growth and oncogenic transformation. Gene amplification of the Her2/neu gene has been observed in a variety of cancer types, including, breast, ovarian, endometrial, gastric, pancreatic, prostate and salivary gland (Hynes and Stern (1994) Biochim Biophys Acta., 1198: 165-184). In breast cancer patients, HER2/neu has also been shown to be of clinical importance as it is associated with poor prognosis, tumor recurrence and shortened survival in breast cancer patients (Seshadri et al. (1993) J. Clin. Oncol., 11: 1936-1942; Berger et al. (1988) Cancer Res., 48: 1238-1243; O'Reilly et al. (1991) Br. J. Cancer, 63: 444-446).
Currently, a great deal of attention has focused on the development of novel immunotherapy strategies for the treatment of cancer. One such strategy is antibody-based cancer therapy. A major goal of antibody-based cancer therapy is to specifically deliver toxic payloads such as radioisotopes, toxins or drugs to tumors. The size range of antibody binding site-based molecules includes: IgM (1000 kDa), IgG (150 kDa), F(ab′)2 (100 kDa), Fab (50 kDa), (scFv′)2 (55 kDa) and scFv (25 kDa). In immunodeficient mice, larger molecules such as IgG and F(ab′)2 fragments are retained at high levels in human tumor xenografts with a low degree of specificity (Adams et al. (1992) Antibody, Immunoconj. Radiopharm., 5: 81-95; Milenic et al. (1991) J. Cancer Res. 51: 6363-6371), while smaller molecules such as scFv, (scFv′)2 and Fab are retained in tumors at comparatively lower levels with greatly improved specificity (Milenic et al. (1991) J. Cancer Res. 51: 6363-6371; Adams et al. (1993) Cancer Res. 53: 4026-4034; Beaumier et al. (1985) J. Nucl. Med. 26: 1172-1179; Colcher et al. (1990) J. Natl. Cancer Inst. 82: 1191-1197).
The most prominent determinant of the above targeting properties is the size of the antibody-based molecule relative to the renal threshold for first pass clearance. Another important feature of antibody-based molecules is valence, as significantly greater tumor retention has been associated with multivalent binding to target antigen (Milenic et al. (1991) J. Cancer Res. 51: 6363-6371; Adams et al. (1993) Cancer Res. 53: 4026-4034; Adams et al. (1996) Proc. Amer. Assoc. Cancer Res. 37: 472; Wolf et al. (1993) Cancer Res. 53: 2560-2565).
Herceptin, a new form of immunotherapy targeting breast cancer, was recently developed to target cancer cells that overexpress Her2/neu. This treatment has been shown in clinical trials to provide effective treatment for patients with HER2/neu positive metastatic breast cancer. However, this drug treatment is costly and is associated with significant morbidity and mortality.
Several other types of therapy have been shown to be more or less effective in breast cancer patients whose tumors express elevated levels of Her2/neu. These include, anthracycline therapy which is thought to be more effective in patients with amplified Her2/neu expression, and hormonal therapy which is less effective in patients whose level of Her2/neu expression is high.
Attention has also focused upon the generation of bivalent single chain Fv-based antibody molecules with molecular weights in the range of the renal threshold for first pass clearance. These include 50 kDa diabodies (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448), 55 kDa (scFv′)2 (Adams et al. (1993) Cancer Res. 53: 4026-4034), 60-65 kDa amphipathic helix-based scFv dimers (Pack et al. (1993) Bio/Technology 11: 1271-1277; Pack (1992) Biochemistry 31: 1579-1584), and 80 kDa (scFv-CH3)2 LD minibodies and Flex minibodies (Hu et al. (1996) Cancer Res. 56: 3055-3061). While each of these proteins is capable of binding two antigen molecules, they differ in the orientation, flexibility and the span of their binding sites. It is believed that these new and innovative immunotherapies will help improve outcomes in breast and other cancers which too frequently recur or progress despite aggressive multi-modality therapy.